Background. Light-emitting diode (LED) phototherapy has been reported to relieve pain and enhance tissue repair through several mechanisms. However, the analgesic effect of LED on incised wounds has never been examined. Objectives. We examined the analgesic effect of LED therapy on incision pain and the changes in cyclooxygenase 2 (COX-2), prostaglandin E2 (PGE2), and the proinflammatory cytokines interleukin 6 (IL-6), IL-1β, and tumor necrosis factor α (TNF-α). Methods. Rats received LED therapy on incised skin 6 days before incision (L-I group) or 6 days after incision (I-L group) or from 3 days before incision to 3 days after incision (L-I-L group). Behavioral tests and analysis of skin tissue were performed after LED therapy. Results. LED therapy attenuated the decrease in thermal withdrawal latency in all the irradiated groups and the decrease in the mechanical withdrawal threshold in the L-I group only. The expression levels of COX-2, PGE2, and IL-6 were significantly decreased in the three LED-treated groups, whereas IL-1β and TNF-α were significantly decreased only in the L-I group compared with their levels in the I groups (p < 0.05). Conclusions. LED therapy provides an analgesic effect and modifies the expression of COX-2, PGE2, and proinflammatory cytokines in incised skin. Low-intensity light therapy, commonly referred to as photobiomodulation, uses light in the far-red to near-infrared region (NIR) of the spectrum 630–1000 nm) and modulates numerous cellular functions. The light-emitting diode (LED), a light source, is as efficient as laser light and can be applied more economically 000 nm an, angiogeSurgical trauma produces injuries in skin, fascia, muscle, and the nerve fibers innervating these tissues and induces subsequent postoperative acute pain. Although many basic and clinical research studies have improved our understanding of the pathologic mechanisms, controlling postsurgical pain well remains a challenge for physicians. The behavioral characteristics of a postoperative pain model in rats produced by hind paw incision resemble postoperative hypersensitivity in humans . The natSince LED therapy can reduce the inflammation response, enhance tissue repair, and relieve pain, this therapy is considered a suitable method for preventing and treating symptoms of tissue damage after trauma or surgery. Thus, the antinociceptive effect of 940 nm wavelength LED therapy on acute pain resulting from tissue injury in a rat incision model was examined and the mechanism of antinociception was explored in this study.Male Sprague–Dawley rats (180–220 g) were used in this study and were purchased from the National Science Council (Taiwan) according to the guidelines for pain research  and the n = 6) after behavioral testing. The staff members who performed the behavioral tests and collected and analyzed the skin tissues were blinded to the animal groups.The rats were randomly assigned to the following groups. Rats received LED therapy 6 days before incision  or 6 days after incision  or from 3 days before incision to 3 days after incision . Three groups of control rats received only a skin incision  and underwent behavioral tests and tissue dissection at 3 hours, 3 days, or 6 days after skin incision for comparison to the L-I, L-I-L, and I-L groups, respectively. Three groups of LED-treated rats received a sham incision and the same LED therapy procedure on the contralateral paw for 6 days and were also considered as control (C) groups. The baseline mechanical withdrawal threshold and baseline thermal withdrawal latency were tested in naïve rats before undergoing a skin incision or LED irradiation, that is, before the incision was made in the I-6 d and I-L groups and before LED irradiation in the other groups. The mechanical withdrawal threshold and thermal withdrawal latency were then tested 3 hours after incision in the L-I and I-3 h groups, 3 days after incision in the L-I-L and I-3 d groups, and 6 days after incision in the I-L and I-6 d groups. Skin tissues dissected from all groups were collected for mRNA and protein analyses  were tested perpendicular to the plantar surface for 5-6 s for each filament. The 50% paw withdrawal threshold was determined using Dixon's up-and-down method . Each th2 of energy density at a power output of 160 mW. The therapeutic procedure began before or after the incision according to the assigned groups and was repeated every 24 hours. The rats were held in the ventral decubitus position to receive LED irradiation. The LED source was kept 1 cm above the injury at a 90° angle. A power checker  was used to check the optical output power of the light source before the beginning of the experimental procedure. The equipment was specially developed for this experiment at the Department of Electronic Engineering of I-Shou University, Taiwan. After LED therapy, the animals were kept in their cages and observed until recovery from anesthesia.The rats in the LED-treated groups received LED irradiation on the left hind paw at a 940 nm wavelength for a period of 30 min to administer 4 J/cmβ-Actin was used as a reference gene. The mRNA amounts of the genes of interest and β-actin were calculated from the threshold cycle (Ct) number. The relative expression level of each sample was normalized to the β-actin expression as an endogenous RNA control. The ΔCt values of the samples were determined as the difference between the Ct of the sample mRNA and the reference gene. ΔΔCt was determined as the difference between the ΔCt of the control groups (C groups) and the ΔCt of the other LED-treated groups. Data were expressed as 2−ΔΔCt to provide an estimate of the amount of sample mRNA present in the LED-treated groups relative to the control group.One microgram of total RNA was used for cDNA synthesis and qRT-PCR gene expression analysis. First, reverse transcription (RT) was performed using a High Capacity cDNA Reverse Transcription Kit according to the manufacturer's protocol . The reaction mixtures were incubated at 25°C for 10 min, 37°C for 120 min, and then 85°C for 5 sec. The final cDNA products were stored at −20°C until use. Next, the cDNA products were amplified by qRT-PCR on a 7500 Real-Time PCR System  using the SYBR® Green PCR Master Mix . The following thermal cycling program was used: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and at 60°C for 1 min. Experiments were performed in triplicate for each data point. The primers used for qRT-PCR are listed in g for 15 min at 48°C. Proteins (30 μg per lane) were separated in a 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane . The membrane was blocked with blocking solution  for 0.5 hours and was rinsed briefly in TBST. The membrane was incubated overnight at 4°C with rabbit anti-PGE2 polyclonal antibody  . A mouse anti-actin monoclonal antibody   was used as a control.Total protein from skin tissue was prepared by the addition 1 : 20 of T-PER Tissue Protein Extraction Reagent   containing protease inhibitors . The tissue was homogenized with a homogenizer. After being placed on ice for 30 min, the homogenate was centrifuged at 10,000β-actin immunoreactivity.After being rinsed with washing buffer for 30 min, the membrane was incubated for 1.5 hours with a horseradish peroxidase-conjugated secondary antibody: goat anti-rabbit IgG-HRP  . The membrane was washed in washing buffer for 30 min, and the antibodies were then revealed using an enhanced chemiluminescence (ECL) detection kit . For densitometric analyses, the blots were scanned and quantified with Image-Pro Plus analysis software , and the results were expressed as the ratio of PGE2 immunoreactivity to t-test. The qRT-PCR data for COX-2, IL-6, IL-1β, and TNF-α and the western blot data for PGE2 were analyzed by the Kruskal-Wallis test to determine differences among groups followed by the Mann–Whitney U test for intergroup differences. The data were expressed as the means and standard deviation. Differences were considered statistically significant at p < 0.05. The statistical analysis was performed with SPSS software .The Kolmogorov–Smirnov test was performed to verify the dependent variables' normality of distribution. The comparison of the mechanical withdrawal threshold and thermal withdrawal latency after treatment and baseline value in each group was analyzed using the paired p < 0.05), but not in the three LED-treated groups    .In the present study, LED irradiation significantly alleviated thermal hyperalgesia in all three LED-treated groups and mechanical allodynia in only the L-I group. LED irradiation also significantly decreased the expression of IL-6, COX-2, and PGE2 in all the treatment groups and decreased IL-1β or TNF-α, in circulating blood 3 hours after carrageenan-induced inflammation. A prior injection of IL-6 antiserum abolished the induction of COX-2 activity and PGE2 release in vascular endothelial cells and attenuated thermal hyperalgesia [A previous study reported an increase in the level of IL-6, but not of IL-1ralgesia , 15. A-nralgesia . Thermalralgesia . Prostagralgesia . Thermalralgesia . Thus, tβ, TNF-α, and IL-6) [α and IL-1β were not suppressed by LED irradiation in the I-L and L-I-L groups. A possible reason why TNF-α and IL-1β were not suppressed by LED irradiation in the I-L and L-I-L groups is that some amount of TNF-α and IL-1β was secreted by Schwann cells that were not inhibited by LED irradiation. LED irradiation was reported to be able to reduce the influx of inflammatory cells to the inflammation site [α and IL-1β, is regulated at local inflammatory sites [β and TNF-α have also been reported to be associated with the development of allodynia and hyperalgesia [The peripheral actions of cytokines in inflammatory pain include the activation of immune cells, such as mast cells, macrophages, and Schwann cells, to produce proinflammatory cytokines  . These cnd IL-6) . We showion site . Howeverry sites , 23, wheralgesia , 25; thiIn addition to being blocked by IL-6, COX activity could be inhibited by LED irradiation. A study by Xavier et al.  showed tHow does 940-nm light therapy inhibit the activation of COX enzymes and decrease the production of PGE2? In a previous study , light iβ and TNF-α 3 hours after the incision, but there was no effect on IL-1β or TNF-α more than three days after the incision. Similar results were reported by previous studies showing that the antinociceptive effects produced by nonsteroidal anti-inflammatory drugs  were the same whether the NSAID was administered before or after the noxious stimulus [Preemptive analgesia is an antinociceptive treatment administered before a noxious stimulus to prevent the establishment of neural nociception processing. Basic physiologic research has created great interest in the potential benefits of preemptive analgesia . Howeverstimulus , 38.β, and TNF-α. Preemptive LED phototherapy transiently suppressed the expression of IL-1β and TNF-α several hours after incision. However, LED phototherapy could not suppress the expression of IL-1β or TNF-α beyond 3 days after incision. This LED therapy holds therapeutic potential for postsurgical pain.In conclusion, the results of the present study demonstrated that 940-nm LED phototherapy could effectively reduce incision-induced thermal hyperalgesia in all the LED-treated groups, regardless of whether irradiation occurred before or after the skin incision, whereas mechanical allodynia was reduced only 1 day after incision. The antithermal hyperalgesic and mechanical allodynic effects of this treatment are probably related to the decreased expression of COX-2, PGE2, IL-6, IL-1


Coordinated shifts of neuronal activity in the prefrontal cortex are associated with strategy adaptations in behavioural tasks, when animals switch from following one rule to another. However, network dynamics related to multiple-rule changes are scarcely known. We show how firing rates of individual neurons in the prelimbic and cingulate cortex correlate with the performance of rats trained to change their navigation multiple times according to allocentric and egocentric strategies. The concerted population activity exhibits a stable firing during the performance of one rule but shifted to another neuronal firing state when a new rule is learnt. Interestingly, when the same rule is presented a second time within the same session, neuronal firing does not revert back to the original neuronal firing state, but a new activity-state is formed. Our data indicate that neuronal firing of prefrontal cortical neurons represents changes in strategy and task-performance rather than specific strategies or rules. Population activity of neurons in the prefrontal cortex has been shown to represent cognitive strategy and rules. Here the authors report that when the same rule is repeated on multiple occasions in the task, it is accompanied each time by a new prefrontal firing rate state. We aim to address the question how a certain behavioural strategy, applied on two different occasions within the same session, is represented in the neuronal firing rate of the prefrontal cortex. The prefrontal cortex is a central structure for executive control of flexible behaviour to assess new rules and strategies, not only in humans5 but also in monkeys7 and rodents10, and is highly interconnected with other brain regions12 indicative of an integrative structure13 and a multifunctional role during cognitive tasks14. The importance of the prefrontal cortex is highlighted by neuronal firing patterns contributing to error-related activity17, working memory23, decision making28, and reward encoding33. It has been shown that lesions of the medial prefrontal cortex lead to an impairment of the ability to follow changing spatial rules34. Prefrontal neurons also change their firing activity when animals are switching between different strategies1. This indicates the importance of the prefrontal cortex for rule-guided behaviour. In addition, behavioural rule changes can lead to abrupt neuronal population activity changes within a very short period of time2. Furthermore, abrupt and coordinated changes of neuronal firing have been reported when animal behaviour reflects uncertainty and evaluation of possible new strategies3. However, the following questions remain unclear: (1) how multiple and consecutive changes of strategy would be reflected in the firing of prefrontal neurons and (2) how the same repeated strategy would be represented on different occasions.In ancient Greek, Heraclitus famously stated that “No man ever steps in the same river twice, for it’s not the same river and he’s not the same man”. He referred to the ambiguity that conscious actions and plans are never truly experienced the same way, however similar they may appear. Earlier research demonstrated how distinct behavioural rules and strategies are entailed in the activity of prefrontal neurons10. In our task design, animals managed to perform under multiple rules during a single recording session, which allowed us to study the neuronal representations when animals changed new rules and acquired new strategies. Additionally, we examined how neuronal population states are changing during the presentation and repetition of multiple rules in the course of a single session.To address neuronal computations in consecutive rule presentations, we used single-unit recordings in freely moving rats to assess neuronal activity during a prefrontal cortex-dependent rule-switching taskThe results of this study show that the neuronal population forms a different and stable firing-state every time a new rule is learnt, even when the same rule is presented twice during the same session. This implies that the concerted neuronal population in the prelimbic cortex does not represent individual rules permanently, but it reflects a change of strategies.n = 5) performed a strategy-switching task with multiple rule changes  within each behavioural session . However, during egocentric rules, the removal or addition of landmarks did not alter performance (p = 0.53)  Fig. . This inn = 95, 54, 74 and 84 for the entire trial, run, reward and inter-trial period, respectively) with either significant negative or positive correlations between their firing rate and task performance  in different trial segments . By examining the firing rate of individual neurons during consecutive trials and different behavioural segments, we observed the following two groups of neurons Fig. : 1) pos posn = 9nts Fig.  indicate38, we divided neurons into two groups: one group of neurons with firing rate higher or equal to 10 Hz, in which an enriched—but not exclusive—population of fast-spiking interneurons is expected, and another group of neurons with firing rates lower than 10 Hz, in which an enriched—but not exclusive—population of pyramidal cells is expected .As fast-spiking GABAergic interneurons might be important for computations involving negative feedback and conflicting resultsted Fig. . When co2. However, the neuronal state dynamics remain unclear when multiple rule changes are presented in the same recording session. As the activity of the prefrontal cells reflects behavioural performance, we tested changes in population activity during multiple rules presentations in a single recording session. For this, each trial was represented as a population vector of neuronal activity: T. For this analysis, we only used trials during learning and learnt phases of the presented rules. We excluded naive phases because they consist of mainly persistent behaviour of the animal still following the previous rule  between observed and shuffled data, when the clusters were defined by the centre of mass. Overall, due to the observation that N-dimensional population vectors of neuronal activity can be clustered, this implies that neuronal activity of the prefrontal cortex holds some form of a representation of rules.The neuronal state representation in the prefrontal cortex changes during rule switchesule Fig. . We obseule Fig. . To test39, which may account for the clustered organisation of our data across consecutive trials and rules. To address this possibility, we performed a multiple linear regression in which we explained the distance between the N-dimensional centres of two clustered rules either by the number of trials (as a measure of time) or by the number of rules separating them. The distance between two rules was calculated by measuring the Euclidean distance between the N-dimensional centres of mass of both clusters. The resulting plane of the regression showed a significant explanation of the distance due to the number of rules in between (p = 2.81e−08) but not due to the trials in between . To further elucidate the contribution of both time and rules to the distance between the rules, two partial correlations were calculated  when the number of rules in-between is taken into account. On the contrary, the partial correlation of the distance of two rules and the number of rules in-between is significant , even when the number of trials in-between is taken into account. Moreover, when Mahalanobis distance was used instead of Euclidean, similar results were obtained. The Mahalanobis distance between clusters is significantly explained by the number of rules (p = 4.756e−05) but not by the number of trials in between the clusters (p = 0.76405). In addition, the interaction term is not significant (p = 0.646). The partial correlations, using Mahalanobis distances, follow the same tendency , Spearman correlation). Overall, this data indicates that the distance of firing vectors between rule clusters is better explained by the presentation of rules per se, rather than by time that has passed between rules, suggesting that rule-dependent switches in strategy may drive the population activity rather than just the passing of time.Nonetheless, it has been shown previously that representations in the prefrontal cortex are drifting over timen p = 2.8e−08 but ted Fig. . The parp < 1e−20, Logistic regression − > p < 1e−20). These analyses suggest that the neuronal firing state during a rule repetition is different from the firing during the initial rule.Having discovered a clustering of neural firing patterns in the prefrontal cortex based on rules, this leads to the question whether those firing patterns could reflect persistent representations of a specific rule. To address this question, we presented the animals with the same rule twice within a single recording session Fig. . Strikinp = 0.58, Wilcoxon rank-sum test and Supplementary Fig. p = 0.248, Wilcoxon rank-sum test), indicating that the repetition of the rule is perceived by the prefrontal cortex’s neuronal population in a similar way as if another new rule was presented.Interestingly, both the Euclidean and Mahalanobis distance between two repeated rules are not significantly different from the distance between two different rule clusters with the same number of rules in between  than those between trials only within ‘A’ with very different trajectories. This implies that the trajectories per se do not explain the separated clustering of the rule repetition.It is known that firing in the prefrontal cortex neurons reflects animal trajectories and movementent Fig. . Differeent Fig. . We compent Fig. . Even foa, b, c are the coefficients of the quadratic equation and ‘s’ is the speed in m/s in a given trial ‘T ’.However, trajectories and speed still might have a more subtle effect. Therefore, we modelled the firing rate of a neuron by using the coefficients of the quadratic equation and the speed of the animal as follows: r = 0.9123, p < 1 × 10−20 and r = 0.9326, p < 1e−20, Spearman correlation).As expected, close to a 30% of the neurons showed a significant correlation to at least one of the coefficients in each of the 4 possible paths (Supplementary Table p = 0.00053) and not the number of trials in between (p = 0.97). In addition, there is not a significant difference between the distances from data of non-repeated and repeated rules .Moreover, projections of the residuals on the first two principal components of the multi-unit activity, still remain clustered Fig.  and ruleOverall, these results support that both the neuronal representations of cognitive rules and the new formation of a neuronal firing state upon the repetition of a rule are not an artefact of the movement of the animals.1. We managed to assess multiple rule changes within the same session, which allowed the possibility to study neuronal population dynamics while following several behavioural changes in strategy. We found two complementary neuronal groups, which dynamically changed their firing during behaviour and their firing rates were significantly correlated with performance. In addition, when multiple rules were presented on the same day, neuronal populations formed new neural states for each rule, even when the same rule was presented twice.To investigate the computations of neuronal populations in the prefrontal cortex during flexible behaviour, we recorded neuronal activity while animals were performing a prefrontal cortex-dependent strategy-switching task41, reward44, encoding of memory and executive functions46 or confidence24. But how do diverse firing patterns adapt when the strategy of an animal changes? Lesions48, optogenetic inactivation49 and pharmacological inactivation10 of the prefrontal cortex have been reported to induce impairment in cognitive flexibility. In the prelimbic cortex, changes in firing rates of single neurons50 and neuronal populations51 have been observed in relation to flexibility in strategy during a rule switching task. Often those changes are presented as a global change of activity and might relate to a complete switch to a different network state, as shown for synchronised changes of neuronal populations during an uncertainty task3. In fact, after lesioning the medial prefrontal cortex, animals could not follow a change of spatial related rules34.Neurons in the prefrontal cortex have a multitude of different firing patterns, which reflect many aspects of the external environment as well as internal computations, including goal-related firing52. This may contribute to the reason why neuronal ensembles in the prefrontal cortex often present abrupt changes in activity whenever a new strategy is behaviourally required2, denoting a flexible state. During the subsequent learning of the new rule, the representation of neuronal responses tends to be more stable and less sensitive to noise53. We show that firing rates of individual neurons are correlated with the behavioural performance of the rat during a rule switching task. We observed two groups of neuron with a different performance-related activity. For one group of neurons, the firing rate was negatively correlated with the performance of the animal, while for a second, complementary group of neuron, there was a positive correlation. Additionally, we also show that these correlations can be found without taking into account the firing information during reward, demonstrating that it is not only a reward-monitoring value of performance. These complementary neuronal assemblies with opposite correlations between firing rate and performance might reflect the simultaneous ability of the network to maintain rewarding behaviours and perform the task with high success or to induce flexible changes when the success rate is low.We assessed how changes in network activity are related to the performance of the animal during multiple rule changes that are presented within the same session, reflecting a high demand on cognitive flexibility. In order to perform a goal-guided behaviour, often the right choice between stability and flexibility has to be found. Thus, the prefrontal network operations should be flexible enough to take into account different sensory inputs and experiences, which provide evidence for a different and more successful strategy, but at the same time should remain stable enough to ignore irrelevant information2, we show that vector populations of trials belonging to the same rule tended to be clustered together, in contrast to vector populations with an intrinsic random organisation. However, by examining the neuronal population dynamics using population vectors, we extended the rule-change analyses to multiple rule changes presented during the same recording session, including in some cases the repeated presentation of the same rule. Not only was each rule reflected by an independent state representation or “rule cluster” different from firing during other rules, but when the same rule was presented again later in that session, the second presentation fell into a new network state, different from the first presentation of this particular rule. Our results suggest that identical rules in a prefrontal cortex-dependent task will not lead to a similar neuronal encoding, but rather to a new set of prefrontal network activity. In line with Heraclitus’s quote, we provide evidence that the same action plan is not “perceived” identically a second time, as the new experiences shaped a new cognitive state. One explanation for this phenomenon might be that firing rates of the prefrontal cortex are context dependent57 and could be influenced not only by past events but also by multiple inputs that the prefrontal cortex is receiving from other areas58. This implies that even when the rule is the same in the second presentation, the actual context in which the rule has been presented differs from the previous presentation. Indeed, due to the fact that time has been shown to be responsible for state changes or drifts in the neuronal activity of the prefrontal cortex39, we decided to test the contribution of time for the new encoding of the repeated rule. Interestingly, when corrected by the number of rules appearing, time was no longer explanatory for the drift , were kept in 12 h light cycle during behavioural experiments (performed during light cycle). All experimental procedures were performed under an approved licence of the Austrian Ministry of Science and the Medical University of Vienna.® 2%) was used before the incision. In order to avoid dehydration, saline solution was injected subcutaneously every 2 h. Seven stainless steel screws were anchored into the skull to improve the stability of the construct and two of the screws were placed onto the cerebellum as references for the electrophysiological recordings. Subsequently, based on the rat brain atlas63, a craniotomy was performed above the prefrontal cortex area , where, after removal of the dura mater, an array of 12 independently movable, gold plated (100–500 kΩ) wire tetrodes  mounted in a microdrive  were implanted. Then, paraffin wax was applied around the tetrode array and the lower part of the microdrive was cemented (Refobacin® Bone Cement) to the scalp. At the end, the surgery site was sutured and systemic analgesia  was given. Animals were given post-operative analgesia (Dipidolor 60 mg diluted per 500 ml drinking water) and were allowed at least 7 days of recovery time.Animals were anaesthetised with Isoflurane  and fixed on a stereotaxic frame, where body temperature was stabilised using a heating pad. Iodine solution was applied to disinfect the surgery site and eye cream was used to protect the corneas. Local anesthetic . The maze consisted of a plus-maze  composed of four arms (80 × 11 cm) with a 90° angle separation between them. Two opposite arms (named North and South) were starting arms and the other two (named East and West) were rewarded arms. The maze was located within four synthetic wall panels to maintain the uniformity of the environment. Three landmarks  were visible and attached to different panels. Reward  was delivered by dispensers (Campden Instruments Ltd). The entire automation of the maze (sensors and pellet feeder controls) was controlled by self-made scripts in MATLAB. Tracking of the rats’ movement was monitored by triangulating the signal from three LEDs  placed on the implanted headstage and recorded at 25 frames per second by an overhead video camera (Sony). One week after surgery, habituation to the maze started. Rats were food-restricted to maintain 85% of their weight. First, during two consecutive days, animals were placed inside of the recording room for a period of around 1–3 h. From the third day on, rats were placed in a ceramic bin at the centre of the maze for an hour and were later positioned on the maze in order to start exploration. Some sugar pellets were placed on the maze and several more at the reward zone. Once the animals learned to explore the maze and consumed the sugar pellets , they were habituated to the activation sound of the pellet feeders, by only giving reward once they entered the reward zone and activated the pellet feeder. After that, the training phase started. It consisted of manually placing the animals at one of the two possible starting points; in this case, the rats had to reach any of the two reward zones, where the reward was given indistinctly. After reward was consumed, rats were manually placed in the bin located at the centre of the maze. Once 30–50 trials were successfully performed by the rat, the full strategy switching task was presented on all subsequent sessions with either rewarded or non-rewarded trials, depending on the actual rule and decision. A typical recording day took place as follows: The rat was kept for 10 min inside of the ceramic bin at the centre of the maze, where baseline recordings were made. Then the task started and the initial rule to be presented was given pseudo-randomly by the script used to control the maze and the task. The animal was taken from the bin and located at one of the two possible starting positions (North or South pseudo-randomly selected) and the bin, where the animal was before was placed to block the opposite starting arm. The rat then ran towards one of the two possible goal arms. After crossing a sensor located at the end of the arm, reward was given in case of being correct. Independently of the correctness of the trial, the animal was taken 2 or 3 s later, placed again in the ceramic bin at the centre of the maze, and after 3–7 s the rat was placed again at one of the two possible starting arms and a new trial was started. The same starting arm could not be selected more than four consecutive times. During the task, when an animal achieved 13 out of 15 possible correct trials, the rule was changed without warning. Four different rules were presented in the task 1: naive ; learning ; and learnt (over 0.6).A strategy switching task based on the consecutive learning of allocentric and egocentric spatial navigation rulesask Fig. : two areThe data presented here correspond to behavioural controls and electrophysiological recordings. Behavioural controls were performed in two animals with a total of 48 rules tested  in 17 sessions performed. The electrophysiological data corresponds to 26 sessions performed by 3 animals . In total, 66 rules changes were achieved (median number of changes per session = 3). For each session, the rules were selected pseudo-randomly (trying to secure at least a switch between strategies and a change of rules within the same strategy). The median number of trials per session is 98.5 trials.64.A headstage  was used to pre-amplify the extracellular electric signals from the tetrodes. Output signals were amplified 1000 × via a 64-channel amplifier and then digitised with a sampling rate of 24 kHz at 16-bit resolution, using a 64-channel analogue-to-digital converter computer card (Axona Ltd). Single-unit offline detection was performed by thresholding the digitally filtered signal (0.8 – 5 kHz) over 5 standard deviations from the root mean square on 0.2 ms sliding windows. For each single-unit, 32 data points (1.33 ms) were sampled. A principal component analysis was implemented to extract the first three components of spike waveforms of each tetrode channel65. Using the Klusters software66, individual single units were isolated manually by verifying the waveform shape, waveform amplitude across tetrode’s channels, temporal autocorrelation (to assess the refractory period of a single-unit) and cross-correlation (to assess a common refractory period across single-units). The stability of single-units was confirmed by examining spike features over time.Spike waveforms from individual neurons were detected using the KlustaKwik automatic clustering softwareAt the end of recordings, animals were anaesthetised with urethane and micro-lesions were made at the tip of the tetrodes by using a 30 µA unipolar current for 10 s . Rats were perfused using saline solution, followed by 20 min fixation with 4% paraformaldehyde, 15% (v/v) saturated picric acid and 0.05% glutaraldehyde in 0.1 M phosphate buffer. Serial coronal sections were cut at 70 µm with a vibratome (Leica). Sections containing a lesion were Nissl-stained to verify the position of the tetrodes.Firing rates were determined in each recording session and for each neuron by dividing the number of spikes over the corresponding time of the trial. Every trial started the moment that the rat initiated movement from the starting arm towards the end arm. The trial can be divided into 3 different behavioural segments , plus another segment which consists on the entire duration of the trial. The run segment was defined as the period from the beginning of rat movement, at the starting arm, towards the reward arm, until the rat crossed a reward sensor located 10 cm at the terminal part of the rewarded area. The reward zone segment was defined as two seconds starting from the moment the animal crossed the reward sensor. The inter-trial segment was defined from the end of the reward segment until the beginning of the next run segment . The entire trial spans from the start of a trial until the start of the next one. Therefore, in total, 4 firing rates were calculated by counting the spikes present during the specific segment and dividing it by the time spent in that segment. The average length (in seconds) for each segment , specified as median, 25th and 75th percentile were: Run , Inter-trial  and the entire trial .mxn, where ‘m’ are the trials and ‘n’ the number of neurons, together with a response vector  RVmx1, shuffle was performed by rearranging the rows of both the firing rate matrix and the response vector, therefore disarranging the temporal organisation of the recording session but keeping the relation between firing rate and responses. The performance was recalculated over this new shuffled data, generating a shuffled performance curve, which was later correlated with the firing rates.Correlation of individual firing rates for each different segment and the entire trial were correlated to the observed performance of the animal using Spearman correlation. In addition, Bonferroni–Holm correction was used to correct for false positives. For the comparison made in Fig. z-scored firing rates of the neurons recorded during that sessionn’ in that specific trial  and Mahalanobis (2) distances were used, ending up with similar results. Euclidean distance is defined as follows:Ta and Tb, defined by the firing rate (FR) of all neurons ‘n’ at that trial Given two trials (or centre of masses of rule clusters) Mahalanobis distance was used mainly between a trial and a rule cluster; therefore, it is defined as follows:n is the mean firing rate of a cell (n) over all the trials conforming the rule cluster, the Mahalanobis distance between the trial and the rule cluster is:Given a trial −1 is the inverse of the covariance matrix.where the operator (‘) is the transpose and SThe similarity index was defined as the normalised Euclidean distance between the coefficients of the quadratic equation modelling a trajectory. The higher the index, the more different are the trajectories.m) can be represented with the coefficients of a quadratic equation:Any given trajectory (TR) in a trial  between two trajectories is defined as:where Clustering was performed using the K-means clustering algorithm. This method requires the number of clusters to be given, and initialisation of the cluster centres (one per cluster to be found). Then, we computed the distance from each point to each centroid and assigned the point to the centroid from which the shortest distance is measured. Thus, a new centroid is calculated from the newly assigned points and the iteration continued until it found a balance (no change in the assignment of new points). For the data in Fig. z-scored firing rate of all the neurons during that trial) to belonging to a given rule ‘A’. The following procedure was done in each recording session, which contained a rule repetition (‘A’ as the rule and ‘A°’ as the rule repetition): Classifiers were trained with a set consisting of random selection of 70% of the trials of rule A, labelled as ‘1’, and the rest of trials of the other rules labelled as ‘0’. Trials of the rule repetition ‘A°’ were not included in the training set. Classifiers were later tested by a data composed of the trials in the rule repetition ‘A°’ and the remaining 30% of trials of rule ‘A’, not used in the training set. Due to the random assignment of trials of rule ‘A’, to both training and test set, the procedure was repeated 1000 times. Figure A logistic regression and a support vector machine with linear kernel were used to create two classifiers which classify a trial  and statistical analyses were performed with MATLAB and Microsoft Excel. All the statistical tests used were non-parametric with Bonferroni–Holm correction unless stated differently in the text. The data used in linear regressions were checked for homoscedasticity. The principal component reduction was performed using a dimensionality reduction toolbox for MatlabThe computer code that supports the findings of this study is available from the corresponding authors upon reasonable request.The data that support the findings on this study are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


The Gram-positive Group A Streptococcus (GAS) is a pathogen that infects multiple tissues in the human host. The virulence regulator Mga in GAS can be phosphorylated by the PTS, affecting Mga activity based on carbohydrate availability. Here, we explored the effects of glucose availability on the Mga regulon. RNA-seq was used to identify transcriptomic differences between the Mga regulon grown to late log phase in the presence of glucose (THY) or after glucose has been expended (C media). Our results revealed a correlation between the genes activated in C media with those known to be repressed by CcpA, indicating that C media mimics a non-preferred sugar environment. Interestingly, we found very little overlap in the Mga regulon from GAS grown in THY versus C media beyond the core virulence genes. We also observed an alteration in the phosphorylation status of Mga, indicating that the observed media differences in the Mga regulon may be directly attributed to glucose levels. Thus, these results support an  In fact, many pathogens have been shown to coordinately regulate the expression of carbohydrate metabolism genes with disease progression during in vivo studies9.Coordinated expression of virulence factors is essential for bacterial pathogens to successfully colonize and elicit an infection in the host. Since expression of virulence genes is often linked to the availability of essential nutrients such as carbohydrates, pathogenic bacteria often utilize carbon catabolism regulatory pathways to sense the presence of preferred carbohydrates and repress the genes involved in alternative sugar utilization1 via two cytoplasmic enzymes, EI (ptsI) and Hpr (ptsH), and several sugar-specific EII components. Each EII comprises two cytosolic subunits (EIIAB), an integral membrane transporter (EIIC), and sometimes a second membrane component (EIID). Phosphotransfer begins when the PEP generated from glycolysis transfers its phosphate to EI, which then is transferred to Hpr on His-15, then a a sugar-specific EIIA, then EIIB and finally to the transported sugar via its EIIC1. When Gram-positive bacteria are in nutrient rich conditions, Hpr is phosphorylated at Ser-46 by the kinase, HprK, allowing HprSer~P to dimerize with the carbon catabolite protein, CcpA, and elicit carbon catabolite repression (CCR) by binding to catabolite response elements (cre) found in promoter sequences10. In the absence of a preferred carbon source, Hpr-His15~P and EIIB~P are capable of phosphorylating histidines within PTS regulatory domains (PRDs) of transcriptional regulators , regulating their activity and the expression of non-preferred sugar operons1. Thus, the PTS represents a signal transduction network through which Gram-positive pathogens alter gene regulation in response to carbohydrate utilization.The phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) allows bacteria to uptake carbohydrates and monitor carbon utilizationStreptococcus pyogenes) is a Gram-positive pathogen that can colonize a variety of tissues in the human host, resulting in both life-threatening invasive as well as benign diseases. Each year around the world, GAS elicits over 700 million self-limiting infections and results in more than 500,000 deaths due to invasive infections and nonsuppurative sequelae11. Both ex vivo and in vivo studies have established that GAS exhibits significant changes in its transcriptome during infection15. As a fastidious fermentative organism that relies heavily on carbohydrates, metabolic genes under CCR are induced in vivo and are often required for full virulence in GAS21, directly linking GAS carbohydrate metabolism and virulence. Here we use the M1T1 strain 5448, a representative of one of the most prevalent serotypes of GAS isolated from invasive forms of infections worldwide.The Group A Streptococcus , including transcription of several sugar transport and utilization operons27. The ‘core’ Mga regulon consists of virulence genes critical for attachment and immune evasion such as M protein (emm), the emm-superfamily , C5a peptidase (scpA), and fibronectin-binding protein (fba)28. The Mga regulon was found to be expressed during the acute phase of GAS-mediated pharyngitis in macaques concurrently with the PTS and sugar metabolism operons15. Taken together, these data underscore that Mga and its virulence regulon are linked to carbohydrate utilization.GAS utilizes global transcriptional regulators such as the ubiquitous stand-alone regulator Mga to coordinate transcriptome changes impacting virulence29. Genetic, biochemical, and structural studies on the homologous Bacillus anthracis toxin regulator, AtxA, indicate that it is also a PCVR33. Two reiterative PRD domains (PRD-1 and PRD-2) of Mga allow PTS phosphorylation to impact its function likely through controlling dimerization of the carboxy-terminal EIIBGat-like domain comparable to AtxA and other Gram-positive sugar-specific PRD-containing activators34. Although it is known that Mga activity is modulated by the PTS, we still do not know whether carbohydrate availability affects Mga-dependent gene regulation. In this study, we explored the effects of glucose availability on Mga and its impact on the Mga regulon. RNA-Seq was used to identify transcriptomic differences between the Mga regulon grown to late exponential phase either in the presence (THY) or absence (C media) of glucose. We observed that Mga was differentially phosphorylated in THY in comparison to C media, which led to a high degree of plasticity of the regulon that is correlated to glucose availability.Mga belongs to a novel family of PTS regulatory domain (PRD)-containing virulence regulators (PCVR) that allows the PTS to directly phosphorylate and affect the activity of Mga based on carbohydrate availability35. Here, we wanted to assess whether the presence or absence of glucose in the environment has an impact on the Mga virulence regulon of the M1T1 GAS 5448. The rich Todd-Hewitt Yeast (THY) media has an initial level of 0.5% glucose (w/v) and was used as a representative of a glucose-rich environment, while C media has a much lower initial level of 0.05% glucose (w/v) and was used to mimic a low- or no-glucose deep-tissue environment35. We directly assayed the change in the concentration of carbohydrates (primarily glucose) in THY  and C media  over an 8-h time course during GAS 5448 growth  and a p value of ≤0.05 were considered significant  were found to be differentially expressed  in C media compared to THY Fig. . Analysii.e., CCR) through directly binding to cre sites under favorable glucose-replete conditions. Since C media contains no glucose at the time point taken, we predicted that CCR would be relieved. 119 (~24%) of the genes differentially expressed in C media , mannitol (spy_1664), galactose (spy_1399), both cellobiose operons (spy_1079–1083 and celB), and trehalose (treCB). The ABC transporters for sialic acid (spy_0213–0215) and cyclomaltodextrin  were also upregulated. Interestingly, the β-glucoside-specific EIIABC and both mannose operons ptsABCD and manLMN were repressed in C media, suggesting they may represent sugar uptake systems for more preferred sugars  sugars were induced during late logarithmic phase growth in C media. These included many alternative PTS carbohydrate EII transporter genes such as those for 3-keto-L-gulonate (has (capsule synthesis) and sag (Streptolysin S) operons; which are also under CCR18. Although there is a considerable overlap between the C media and CcpA regulons, there were also media-specific regulatory phenotypes. An additional 25 genes found in this study have been previously shown to be regulated by CcpA18, but were regulated in a different direction. Interestingly, we observed a significant increase in the expression of the lacD.1 operon and rgg/ropB, both of which encode for regulators of the secreted cysteine protease virulence factor gene speB. C media is an inducer of speB expression in M14 GAS35; however, we did not see speB being differentially expressed in M1T1 GAS 5448. We also found several genes of the core Mga virulence regulon to be down regulated in C media  and the C5A peptidase (scpA) were down regulated.Several virulence-related genes were upregulated during growth in C media, including the mga itself, is also reduced in C media compared to a glucose-rich environment.Taken together, the transcriptome of M1T1 GAS 5448 in C media exhibits similarity to the release of CcpA-mediated repression likely due to the absence of glucose in C media at late logarithmic phase. Additionally, expression of some of the Mga regulon, but not mga itself, we hypothesized that the Mga regulon in these two medias would vary. An insertional inactivation of mga in M1T1 5448 was constructed using a temperature-sensitive plasmid to generate the mga mutant, 5448.930 (see Methods). Growth kinetics of 5448.930 were found to be comparable to WT 5448 in both THY and C media (data not shown). Total RNA was isolated in two biological replicates from the WT 5448 and the ∆mga mutant grown in either THY (+glucose) or C media (low glucose) to late logarithmic phase and processed for RNA-seq as described in Methods. Data obtained from the WT 5448 was compared to the ∆mga grown cells in either media (WT/∆mga) with changes in gene expression over 1.8-fold (log2 ≤ −0.90 or ≥0.90) and a p value of ≤0.05 considered significant , showing between 7-fold to 18-fold reduction in transcript levels in the ∆mga mutant (Table S4). Interestingly, we did not observe a Mga-specific phenotype for sclA in our RNA-Seq datasets, a gene considered to be part of the core Mga regulon39. The mga mutant 5448.930 was passaged at the permissive temperature to generate the 5448.930R rescue strain as a complement control  encoded in the GAS genome, the second of which is not regulated by Mga.The most activated genes in both media conditions were those of the core Mga regulon  were significantly regulated by Mga with approximately equal numbers of repressed (73) and activated (75) genes , exhibited 4-fold lower transcript in the mutant compared to WT 5448  and its inhibitor (spi) in the M1T1 5448 background in THY  that were regulated by Mga  in C media, representing 7.2% of the GAS genome  was also activated by Mga. In addition, the phage-encoded streptodornases sdaD2 (Sda1) and spd3 (Spd-3) were also regulated by Mga in C media, with both exhibiting ca. 5-fold reduction in a ∆mga mutant  that could be complemented upon glucose supplementation, indicating that Mga regulation of this streptodornase occurs in a glucose-dependent manner. In support of this finding, E64-treated (cysteine protease inhibitor) supernatants from ∆mga mutants grown to late logarithmic phase in C media exhibited a decrease in steady-state Sda1 levels compared to WT 5448 . As with THY, the chromosomally-encoded sagA-H), Streptolysin O (slo), Streptokinase (ska), Streptococcal secreted esterase (sse), the heme utilization operon (hupYZ), and the osmotic stress operon (opuAA/opuABC) were regulated by Mga only in the glucose-depleted C media , mac (IgG-degrading protease), spd , M5005_spy0522 (sugar hydrolase), M5005_ spy0143 , and spxA.2 . Despite the stark difference in genes that are regulated by Mga in a glucose versus a no glucose condition, the broad COG categories represented were not different. In THY, the top three categories of Mga-regulated genes were unknown function , carbohydrate utilization , and defense mechanism/virulence   in comparison to THY (+glucose) Fig. . We wereTHY Fig.  and emm,speB in THY at late log phase  the majority of the changes appear to be specific to glucose availability, and (2) some changes in the regulon dynamics are due to C media-specific differences.Mga was shown to repress the transcription of mga itself was not affected (Table 3 an M4 ∆mga (79% identity to M1 Mga) GAS strain (KSM547) that expressed a His-tagged version of either WT Mga (pKSM808) or a phosphoablative A/A/A Mga (pKSM871) that substitutes all three PTS-phosphorylatable histidines with alanines from a replicating plasmid (see Methods). The two strains were grown in either THY or C media to late log growth, whole cell lysates were isolated, and separated by Phos-tag SDS-PAGE for Western blot using α-Mga4 antibodies. The blot revealed two bands for WT 5448 lysates grown in THY, a slow-migrating more diffuse upper band arising from phosphorylated Mga at one or several of 3 potential histidines  and a larger fast-migrating band corresponding to non-phosphorylated Mga . In C media at a point where glucose is depleted, the slow-migrating Mga4~P-His6 is reduced, indicating less PTS phosphorylation  compared to C media (low glucose), yet ed Table . Since Mion Fig. . As wouldia Fig. . The ratach Fig. . This shach Fig. . While aemm and mga itself. There was a strong correlation between the genes activated in C media (low glucose) with those known to be repressed by CcpA, indicating that C media relieves CCR. Interestingly, when comparing the Mga regulon in both medias, we found very little overlap beyond the core virulence genes, despite regulating similar functional categories (COGs). Many of these differences in Mga-regulated genes between the two medias could be directly attributed to glucose levels, which led to an alteration in the PRD-mediated phosphorylation patterns of Mga. Thus, Mga and its regulon are likely influenced by glucose and further support an in vivo link between glucose availability and virulence regulation in GAS.The current study was undertaken to assess how glucose affects global transcriptional changes in the GAS cell through Mga. Our RNA-seq results revealed that in the WT M1T1 5448 some of the Mga regulon is up regulated in THY (+glucose), but not cre) sites. Metabolic genes that enable the utilization of alternative (non-glucose) sugars are classically induced in the absence of the preferred carbohydrate source, glucose. For GAS, two studies have determined the genome-wide, CcpA-induced regulons in M1T1 serotype18, which found that ~8% of the GAS genome was regulated by CcpA in THY. Here, we observed that 16% of the GAS genome was differentially expressed when grown in the absence of glucose (C media at late log phase), which overlapped with the published CcpA regulons18. Thus, it appears that the lack of glucose encountered by GAS in C media at this point in growth is capable of relieving CCR and stimulating the transcription of alternative carbohydrate uptake systems. Although there are clear commonalities, our data also showed that GAS undergoes a much broader transcriptomic change during growth in C media  when compared to growth in THY  is controlled by the master regulator, CcpA, through direct binding to catabolite response elements  grown to late-logarithmic growth in THY, involving ~10% of the GAS genome that encompassed mostly virulence and carbohydrate metabolism genes27. Recently, RNA-seq of Mga-regulated genes in an M59 (mga-2) serotype, also at late-logarithmic growth in THY, revealed ~7% of the genome was involved, primarily related to virulence, metabolism, and unknown functions29. Our RNA-seq results revealed that the Mga regulon in an M1T1 GAS strain under similar growth conditions regulated ~8% of the genome, in line with the recent M59 regulon. Although the M1 (SF370) and M1T1 (5448) strains analyzed differ by only 7% in their genomes (mostly prophage content), comparison of the Mga  regulon datasets revealed very little overlap. A similar trend was observed when regulons from strains expressing two mga-2 alleles (M59 and M4) were compared29. We saw that SF370 and 5448 shared only 8 Mga-regulated genes that were regulated in the same direction, with 5 genes being members of the core Mga-regulated virulence genes , although the degree of activation appears to vary29.The Mga regulon was initially determined by our group based on microarray analysis of 3 diverse serotypes representing both lacG , lctO (lactate oxidase), and M5005_spy0143 . Both lacG and lctO are involved in lactose metabolism. LctO converts lactate into pyruvate and leads to production of hydrogen peroxide (H2O2) in a growth phase- and glucose-dependent manner through direct repression by CcpA19. In S. pneumoniae, increase of H2O2 production acts as a virulence trait that aids in colonization by slowing the clearance of bacteria through the inhibition of ciliary beating in the upper respiratory tract and by promoting bactericidal activity to outcompete other microbial species from the microflora44. Therefore, it’s possible that Mga activates the transcription of lctO in order to up regulate hydrogen peroxide production just enough to aid in colonization, which is the stage of infection that correlates with the highest level of Mga activation15. Interestingly, M5005_spy0143 encoding a small hypothetical protein was also found to be regulated in C media  were both observed as being repressed by Mga in M1T1 5448 grown in THY  DmgB, both of which are homologs of Mga24. Therefore, a potential link between bicarbonate transport and Mga gene regulation may exist. Since the number of genes that overlap between the THY and C media Mga regulon are so few, we propose that this set of nine novel genes may also be a part of the core M1T1 Mga regulon.In both THY and C media, Mga regulates the same approximate number  of the non-prophage GAS genome and representing comparable COG categories Figs  and the sda1and spd3) were activated by Mga in C media  in vivo51. It has been suggested that in the covS+ M1T1 MGAS2221, the Mga regulon, not Sda1, plays a much more important role in the covS switch in vivo52. It may be that during invasive infection with covS+ M1T1 strains, a glucose-deplete environment leads to Mga-dependent activation of sdaD2 (and other streptodornases) that impacts the covS switch.Both M1T1 phage-encoded DNases  in C Media (Table emm was not observed. Two possibilities for the reduction of Mga regulon expression in C media compared to THY are: (1) CcpA has been shown to bind to a cre site located upstream of the P1 promoter region of mga and stimulates transcription53; therefore, in the low-glucose environment of C media, CCR is relieved, causing a decrease in expression of the Mga regulon under a low glucose condition. Alternatively, (2) PTS-mediated phosphorylation of Mga activity may impact regulation of the core genes29. The latter scenario is the most probable, as the transcript levels of mga were not significantly different between THY and C media, indicating possible posttranslational modification. The lack of differential emm expression is not clear at this time and we are exploring this mechanism further.We observed a 4- to 5-fold repression of some core Mga regulon genes  would result in an active non-phosphorylated Mga and remain active. Hence this would help explain the activation of the regulon in THY over C media  no phosphorylation produces an active Mga, (2) phosphorylation on only PRD2 can cause an increase in activity, and (3) phosphorylation on PRD1 inhibits Mga activity, and is dominant over PRD2 phosphorylation no phospet al.3, since glucose is initially present in both THY and C media. Further, since RNA was isolated from strains grown to late log, there still may be a small but significant amount of glucose present in C media to maintain the phosphorylation status of Mga. Regardless, we now believe it may not only be the level of glucose that is important for Mga activity, but the mere presence of it during growth which could affect the phosphorylation status of Mga. The unphosphorylated band for the A/A/A Mga in both C media and THY indicates that phosphorylation by the PTS occurs at the phosphohistidines on the PRD domains of Mga.Surprisingly, Mga from GAS grown in THY showed two bands on a Phos-tag gel representing a phosphorylated and unphosphorylated species. In C media, far less of the phosphorylated form of Mga was observed, while the phosphoablative (A/A/A) construct was unphosphorylated under both media conditions Fig. . TherefoThis work shows that glucose directly affects Mga activity and the composition of the Mga regulon. Furthermore, the differences between the regulons in the presence or absence of glucose is most likely attributed to a phosphorylation event that occurs on the phosphohistidines located on the PRD domains of Mga. While it is unclear which phosphohistdine(s) are affected and the mechanism by which Mga is able to regulate genes alternatively, it is clear that in the presence of glucose, the PTS plays an important role in activating this global transcriptional regulator and virulence genes downstream.Streptococcus pyogenes (GAS) strain M1T1 544854 is isolated from an invasive infection. The reference genome used in this study was generated from the M1T1 strain MGAS500555. GA40634 is a clinical isolate of the GAS M4 serotype and SF370 is a sequenced M1 strain. KSM547 and KSM165-L are isogenic strains of GA40634 and SF370, respectively, with an insertional inactivation of the mga gene38. GAS bacteria were either cultured in Todd-Hewitt medium supplemented with 0.2% yeast extract (THY) or in C Media56 buffered to pH 7.5 with NaOH. A 25% (w/v) stock solution of glucose was used to supplement C media to the levels indicated in select experiments. GAS growth was assayed via absorbance using a Klett-Summerson colorimeter (A filter) and expressed in Klett Units.Escherichia coli (E. coli) strain DH5α (hsdR17 recA1 gyrA endA1 relA1) and C43[DE]57 were used as the host for plasmid constructions, and C41[DE3]57 was used for protein expression, as indicated in Table E. coli strains were grown in Luria-Bertani (LB) broth or ZYP-505258 for protein expression. Antibiotics were added to media, as needed, at the following concentrations: spectinomycin at 100 µg/ml for both E. coli and GAS, kanamycin at 50 µg/ml for E. coli and 300 µg/ml for GAS, and 100 µg/ml of ampicillin for E. coli.E. coli using the Wizard Plus SV miniprep system (Promega). DNA fragments were agarose gel purified using the Wizard SV gel and PCR cleanup system (Promega). PCR for generating probes and cloning was conducted using Accuprime Pfx (Life Technologies) according to the manufacturer’s recommended protocol. PCR for diagnostic assays was performed using Taq DNA polymerase (NEB). Genewiz, Inc performed all DNA sequencing. The Master-Pure complete DNA and RNA purification kit for Gram-positive bacteria  was used to extract genomic DNA.Plasmid DNA was extracted from mga from 5448 gDNA, which was amplified using the primers Mga InIn F and Mga InIn R  were identified following selection with spectinomycin and growth at the non-permissive temperature (37 °C). PCR on isolated gDNA was used to confirm plasmid insertion for each GAS mutant using primers SPR1 and SPR2  transposon mutants generated in 544859. Library #16 was screened using AP-PCR60 followed by sequencing and alignment of sequence to the 5448 genome. Transposant #1 (16.1) was mapped to the 5′-end of mga in the sense direction.A transposon insertion in 21. Briefly, Direct-zol RNA MiniPrep kit (Zymo Research) was used to isolate total RNA using a modified procedure to improve GAS cell disruption. Frozen cell pellets were resuspended in 700 µl of Trizol plus 300 mg of acid-washed glass beads (Sigma Life Science) and disrupted by vortexing for 5 min. RNA samples were DNase-treated using the Turbo DNase-free kit (Life Technologies). A total of 5 µg of this RNA was treated for ribosomal RNA (rRNA) removal using the Ribo-Zero Magnetic kit for Gram-positive bacteria (Epicentre). Quality and quantity were assessed using a 2100 Bioanalyzer (Agilent) NanoDrop 8000 spectrophotometer (Thermo Scientific), respectively. Directional RNA-Seq libraries were created using the ScriptSeq v2 RNA-Seq Library Preparation kit (Illumina) according to the manufacturer’s recommendations.RNA sequencing (RNA-Seq) was performed as previously describedA rapid-run 100 bp single-read DNA sequencing was then performed at the Institute for Bioscience and Biotechnology Research (IBBR) Sequencing Facility at the University of Maryland, College Park, using the Illumina HiSeq 1500 platform. Data were generated in the standard Sanger FastQ format and raw reads were deposited with the Sequence Read Archive (SRA) at the National Center for Biotechnology Institute (accession PRJN412519).61, filtered and trimmed using trimmomatic62 and mapped against the MGAS5005 genome (accession CP000017) using alignment software, as previously described. Differential expression analyses were performed following size-factor and quantile normalization of read counts. Limma63 and DESeq64 statistical models were used to account for batch effects. The resulting metrics of expression were visualized using Circos65, and tested for ontology enrichment using a variety of ontology software packages .Read quality was measured using FastQC21. Briefly, DNase-treated total RNA from strains were added to SYBR green master mix (Applied Biosystems) with 6.5 µl of each gene-specific real-time primer from a 20 nM stock (Table http://biotools.umassmed.edu/bioapps/primer3_www.cgi).The quantitative  reverse transcription-PCR (qRT-PCR) experiments were performed as previously described21, using cDNA generated separately in a two-step protocol in order to take into account the strand-specificity of the RNA-Seq results.RNA-Seq data validation was performed as previously describedgyrA transcripts, which acts as an internal control. Standard error was calculated from three biological replicates, and differences over 2-fold in expression were considered significant. Correlation coefficients were determined by graphing the log value of the RNA-seq result on the X-axis to the log value of the RT-qPCR on the Y-axis. The R2 value was calculated using a linear regression model, which represented the fitness of the data . Although typically used to monitor blood sugar, we used this apparatus to monitor glucose concentration of THY, CDM, and whole human blood during GAS growth. Values are given in mg/dl. Readings were taken by first inserting a test strip (AimStrip® Plus) into the monitor, placing the test strip into the culture to draw up liquid droplets, and waiting for 10 seconds for the final reading.6 was purified as previously described34. Briefly, E. coli containing the plasmid pMga1-His was grown in ZYP auto-induction media for ~50 h at 37 °C. Cells were harvested by centrifugation at 4 °C. Pellets were then resuspended in Lysis Buffer A  and 1× EDTA-free cOmplete protease inhibitor (Roche), and incubated on ice with lysozyme for 30 min followed by sonication using a Branson Sonifier 450 with a tapered microtip  pulsing 3 × 1 min with 3 min breaks on ice between cycles. The lysate was spun for clarification at 20,000 × g for 30 min at 4 °C 3–4 times. The lysate was loaded on a 5 ml NiNTA agarose resin (Qiagen) and rotated at 4 °C for 1 h. The column was then washed twice with NiNTA Wash Buffer . Protein was eluted with 10 ml of NiNTA elution buffer  and chosen fractions were stored at −20 °C. Protein concentration was analyzed using the Bio-Rad protein assay kit, reading the absorbance at 595 nm on a spectrophotometer.GAS Mga1-His34. Briefly, GAS cells were inoculated 1:20 into 12 ml of THY or C media56, grown to late-log, and pelleted by centrifugation for 15 min at 6,000 × g. Pellets were resuspended in 200 µl of Buffer D  glycerol), 1 × cOmplete EDTA-free protease inhibitor (Roche), 10 µl (20 U) of Turbo DNase (Life Technologies), and 5 µl (250 U) of PlyC71. The cells were mixed by flicking and incubated on ice for 20 min, followed by centrifugation at 13,000 × g for 10 min at 4 °C. Clarified supernatants containing soluble proteins were extracted and boiled with 3 × cracking buffer  at 95 °C for 5 min, and stored at −20 °C. Protein concentration was assayed using the Bio-Rad protein assay kit, reading the absorbance at 595 nm on a spectrophotometer.Soluble GAS protein extractions were performed using the bacteriophage lysine, PlyC (kindly provided by D. Nelson) as previously describedSelected protein samples were run on a 10% SDS-PAGE, with 6% stacking gel, for 1 h at 180 V. The gels were then transferred to nitrocellulose membranes using the Mini-Protean apparatus (Bio-Rad) in 1X transfer buffer . Membranes were blocked overnight at 4 °C in blocking solution (5% (w/v) dried milk in PBS-tween). Primary antibodies were incubated at a 1:1,000 dilution, unless otherwise stated. The following primary antibodies were used at a 1:1,000 dilution: α-His antibody (Roche), α-Mga4, α-Mga1-HRP, and α-Sda1 . Blots were then washed three times for 5 min in PBS-tween. The primary antibody Hsp60 (Enzo Life Sciences) was used at a 1:2,500 dilution. Blots were then incubated with secondary antibodies: α-rabbit-HRP  or α-mouse-HRP  for 1 h. The blots were then washed with PBS-tween three times for 5 min, and visualized using SuperSignal West Femto Substrate (Thermo Scientific) and a LAS-3000 CCD camera (FUJIFILM).2+-Phos-tag 10% SDS-PAGE gel (WAKO), with 6% stacking gel. The resolving gel was made as follows: 1.75 ml of 1.5 M Tris-HCl, 1.75 ml of 40% bis-acrylamide, 140 µl of 10 mM MnCl, 140 µl of phos-tag solution, 3.1 ml of dH2O, 100 µl of 10% ammonium persulfate, and 20 µl of TEMED (Bio-Rad). Protein samples were run for 2 h at room temperature and 2 h on ice, for a total of 4 h at 180 V, prior to western blot as described above.The phos-tag gel system was used to visualize the phosphorylation state of Mga. Briefly, GAS whole cell lysates were run on a MnThe authors have deposited all RNA-seq raw sequencing reads with the Sequence Read Archive (SRA) at the National Center for Biotechnology Institute (accession PRJN412519) for public availability.Supplementary Materials


Copper, which can potentially be a highly toxic agent, is an essential nutrient due to its role as a cofactor for cuproenzymes and its participation in signaling pathways. In mammals, the liver is a central organ that controls copper turnover throughout the body, including copper absorption, distribution, and excretion. In ontogenesis, there are two types of copper metabolism, embryonic and adult, which maintain the balance of copper in each of these periods of life, respectively. In the liver cells, these types of metabolism are characterized by the specific expression patterns and activity levels of the genes encoding ceruloplasmin, which is the main extracellular ferroxidase and copper transporter, and the proteins mediating ceruloplasmin metalation. In newborns, the molecular genetic mechanisms responsible for copper homeostasis and the ontogenetic switch from embryonic to adult copper metabolism are highly adapted to milk ceruloplasmin as a dietary source of copper. In the mammary gland cells, the level of ceruloplasmin gene expression and the alternative splicing of its pre-mRNA govern the amount of ceruloplasmin in the milk, and thus, the amount of copper absorbed by a newborn is controlled. In newborns, the absorption, distribution, and accumulation of copper are adapted to milk ceruloplasmin. If newborns are not breast-fed in the early stages of postnatal development, they do not have this natural control ensuring alimentary copper balance in the body. Although there is still much to be learned about the neonatal consequences of having an imbalance of copper in the mother/newborn system, the time to pay attention to this problem has arrived because the neonatal misbalance of copper may provoke the development of copper-related disorders. A suboptimal content of micronutrients in a mother’s diet during pregnancy and lactation may be a cause of developmental defects in newborns. The data show that severe neonatal defects can be prevented by correcting a mother’s diet  ,5,6,7,8.In newborns, the mechanisms for excreting copper through bile and controlling copper absorption in the small intestine do not operate . TherefoCopper is an essential component of the cells of almost all modern organisms. It was involved in the metabolic processes in the early stages of the Earth’s evolution, possibly at the beginning of the Great Oxygenation Event, when it became bioavailable due to the oxidation of insoluble sulfides . In aqueThe native tertiary structure of cuproenzymes is formed during the successful metabolic insertion of copper ion(s). The removal of copper from holoenzymes in vitro or defects in the processes of the metalation of apo-enzymes in vivo can cause the disruption of the tertiary structure and the loss of catalytic functions . TherefoIn the past few years, evidence of the existence of a regulatory role of copper has accumulated. This role has remained elusive for a long time, although the first evidence for the involvement of copper in the regulation of endothelial cell growth was obtained almost 40 years ago . RecentlTwo prerequisites are implied from the existence of copper regulatory functions: (1) a pool, in which copper is accumulated and from which it is liberated on demand, and (2) copper-regulated sensors. In cells, two copper pools that may take part in the rapid, local change of copper concentrations exist .The first pool is a system that includes metallothionein-Cu(I), glutathione (Cu(I)/Cu(II)), and COMMD1-Cu(II) (Copper Metabolism gene MURR Domain 1), which possibly controls the changes of the copper concentration in the cytosol . The mem64Cu is taken up through the basolateral membrane Cp and [125I]-Cp pass on [64Cu] into the cytosol, and the protein moiety (apo-Cp) is absorbed by endocytosis, desyalated in endolysosomal vesicles, and then returned into circulation. This Cp with a processed carbohydrate moiety is then captured by endocytosis in hepatocytes through the group-specific receptor of acidic desalted glycoproteins Cp into the milk [14C]-Cp was measured. The mRNA coding the secretory form of Cp is found in the transcriptome of the cells of the lactating mammary glands. The length of milk Cp-mRNA and the molecular mass of milk Cp do not differ from hepatic Cp-mRNA and plasma Cp correspondingly [In adult mammals, copper assimilated in the small intestine is typically absorbed completely by the liver in several minutes. In approximately 90 min, copper is returned to the bloodstream as a component of serum Cp ,137. Howthe milk . This haondingly ,141. Milondingly ,143,144.ondingly ,143,144.Cp gene, as well as the Ctr1 and Atp7b genes, which provide Cp metalation, are observed shortly before the end of gestation [Cp, Ctr1, and Atp7b genes gradually decrease according to the change in the Cp concentration in the milk. The same pattern of Cp gene expression was reproduced in vitro in the PMC42-LA mammary epithelial cell culture models [In the mammary gland cells, strong and rapid upregulation of the estation . During e models . The acte models ,148,149.Cp gene (at position—1966). This nucleotide is part of the cis-element for transcription factor C/EBPβ (CCAAT/enhancer-binding protein beta), which may potentially take part in the gradual suppression of the Cp gene activity [Cp gene in breast cells during lactation has been preserved by natural selection.In milk, copper is present in a nondialyzable fraction, and approximately 75–80% of copper is found in Cp . In the activity . In Turkactivity . In all activity ,154,155.−/−Cp gene in mice causes a decrease in the copper concentration in the milk of females and copper misbalance in the pups [Atp7b gene in the toxic milk line of mice [tx/tx mice dies because of the copper deficiency in the mother’s milk, but it completely survives if fed from the first day of life by a wild-type ‘nurse’ [The high biological importance of milk Cp is demonstrated by the following facts. Copper atoms that are associated with Cp molecules are assimilated by newborns more easily . The knothe pups . The sam of mice . The pro ‘nurse’ . In the  ‘nurse’ , perhaps ‘nurse’ . A speci ‘nurse’ ,160. It It is worth nothing that the data discussed in this section were obtained from rats. Although the infant liver is different from the rat pup liver with respect to some species-specific features, the facts obtained for rodent copper metabolism are valuable for humans. In both species, at the ETCM stage, the copper status (low copper and ceruloplasmin concentrations in the serum), the copper accumulation in the liver tissue, the excretion of copper with urine but not with feces, and the switching to the ATCM , as well as the dynamics of the decrease in the copper and Cp concentrations in milk during lactation, are similar. These data allow us to determine that during development, the expression of the copper metabolism genes in the liver and mammary gland develop similarly in rats and humans.2Cu(II) complex. In infant formula, the copper is dialyzable, it is not “packed” into Cp, and its concentration in the diet from birth up to 6 months of age does not change. Therefore, newborns fed with such formulas receive copper in a highly mobile form, which easily gets to the bloodstream but is not delivered to the endosomes of hepatocytes. Additionally, the daily copper supply progressively increases along with the volume of consumed food. By the end of the first month, the copper consumption of formula-fed infants exceeds the normal copper consumption of breastfed infants by many times [Children exclusively fed cow milk, which contains copper and milk Cp but at concentrations that are lower than those of human milk, at an age of 6 months develop severe anemia, neutropenia, hypocupremia and display malformation of their bones and defects in erythrocyte maturation and so on . These sny times . In ratsny times ).Cp gene at the transcription and translation levels. The copper status was also affected in the cerebrospinal fluid. The concentration of copper and Cp was increased by a factor of 7. The specific content of copper in the brain cells did not change. In newborn rats with ETCM and ATCM, which were supplemented with copper ions, the Ctr1, Atp7b, and metallothionein mRNA levels in the liver increased [The transition manifests as an abrupt drop in the copper concentration in the liver, an increase in the blood serum copper levels, and preterm activation of the ncreased . Simultancreased  and coppncreased  showed tncreased .Milk proteins are divided into two groups: nutritive proteins, which are used for growth, and bioactive proteins, which perform regulatory and transport functions . In milkThe reviewed data suggest that the copper imbalance in the early postnatal period, which is induced by feeding infants formula, influences various aspects of copper metabolism. This is primarily an increase in the Cp and copper concentrations in the blood serum and cerebrospinal fluid, but there is no evidence that it can affect the formation of cuproenzymes. It is possible that, in individuals that carry no latent inherited defects in copper homeostasis, the nutrient copper excess is compensated in further development and has no significant impact on health. However, the individuals carrying heterozygous mutations in genes related to copper homeostasis  may be especially sensitive to copper imbalance in early childhood . The disThe delayed effects of the impairments of copper homeostasis in early infancy remain poorly studied. If this problem is ignored, there is a risk that it will impact the development of intellectual abilities and physical and mental health. Although many aspects of copper metabolism need further thorough investigation, it may be stated that for the ideal development of the intellectual and physical qualities of the individual, significant attention should be given to the balanced content of copper in the diet of infants.


It has been shown that near-perfect thermal camouflage can be continuously achieved for background temperatures ranging from 30 °C to 50 °C by tuning the emissivity of the device, which is attained by controlling the GST phase change. The thermal camouflage is robust when the observation angle is changed from 0° to 60°. This demonstration paves the way toward dynamic thermal emission control both within the scientific field and for practical applications in thermal information.Camouflage technology has attracted growing interest for many thermal applications. Previous experimental demonstrations of thermal camouflage technology have not adequately explored the ability to continuously camouflage objects either at varying background temperatures or for wide observation angles. In this study, a thermal camouflage device incorporating the phase-changing material Ge Thermal camouflage surfaces developed by Chinese researchers can be tailored to hide objects in front of different backgrounds. Traditional thermal camouflage comprises low-emissivity cloaks that lower the apparent temperature of vehicles or people to match their surroundings; however, objects can only be well-hidden when the background is one particular temperature. Qiang Li and co-workers at Zhejiang University in Hangzhou deposited a germanium-antimony-tellurium alloy onto gold film, before thermally annealing their samples at 200 °C. By varying the annealing time, the researchers prepared samples that were completely amorphous (randomly structured), completely crystalline (ordered), or intermediate states between the two. They found that the more crystalline samples had higher apparent temperatures, meaning the surfaces could be tailored to work at a range of background temperature. As well as benefitting military, such technology could allow better heat management during space travel. In terms of the physical mechanisms behind it, camouflage technology can be divided into two categories: color camouflage and thermal camouflage. Color camouflage is achieved by tuning light reflection or transmission to match the object’s appearance to the background in the visible or near-infrared range4. Many natural creatures such as cephalopods or chameleons can rapidly change their skin color to camouflage in this way6. The function of thermal camouflage, however, is to match the radiation temperature of a given object Tr (defined as the temperature at which a blackbody has an integrated radiance Bbb(Tr) equal to that of the object at temperature T, Bbb(Tr) = B(T)) with that of the background by controlling thermal emission. Therefore, thermal camouflage can befool the thermal imagers, which distinguish the object from the background based on their radiation temperature difference. Traditional thermal camouflage uses a low-emissivity coating, which reduces the object’s radiation temperature, to make the object blend into the background7. However, the emissivity of a traditional camouflage coating is fixed, and the objects can only be camouflaged in a fixed background temperature. Once the background temperature changes, the object can be easily discovered by the thermal imagers because of the radiation temperature difference between the object and the background. In addition, the observation angle robustness of thermal camouflage is an important factor for practical applications, as the targets can be discovered from wide observation angles. Therefore, thermal camouflage technology that can be used at varying background temperatures and wide observation angles needs to be explored.Camouflage technology, which has the ability to hide or disguise an object  in the background24 and contribute to a number of applications, including radiative cooling28, thermophotovoltaics35, and sensing37. Recent works have extended emissivity control to dynamically reconfigurable materials, including graphene38, quantum wells39, doped zinc oxide40, and thermochromic metamaterials41. However, these works have not investigated their potential uses for creating thermal camouflage. It has, however, been demonstrated that the thermal emission of a device consisting of vanadium dioxide (VO2) can be controlled dynamically and it can therefore be used for thermal camouflage43. Moreover, thermal camouflage for a specific background temperature distribution can be created using natural materials44 or metamaterials based on transformation thermotics45. However, the ability to continuously camouflage objects either at varying background temperatures or for wide observation angles has not yet been experimentally explored.Thermal camouflage with the ability to tune the thermal emissivity of the object can be used at varying background temperatures. Most of the methods to engineer emissivity and thermal emission are static2Sb2Te5 (GST) film on top of a metal film is experimentally demonstrated. The phase-changing material GST has been used in various switchable optoelectronic devices62. Here we report the first thermal camouflage device incorporating the phase-changing material GST, which can camouflage at varying background temperatures and for wide observation angles. Continuous thermal camouflage is achieved by controlling the GST phase transition, during which the imaginary part of the permittivity of the GST increases, which, in turn, increases the emissivity. This thermal camouflage device presents several advantages as follows: (1) large background camouflage temperature range: thermal camouflage is experimentally demonstrated when the background temperature changes from 30 °C to 50 °C. (2) Robust with regard to the observation angle: the thermal camouflage is robust over a large range of observation angles, from 0° to 60°. (3) Size flexibility: the emissivity of the surface can be spatially tuned with a patterned layer of GST. We have demonstrated that the spatial emissivities of patterned GST-Au samples exhibit a spatial resolution of 500 μm (checkerboard pattern) and 100 μm (ZJU pattern). (4) Easy fabrication: the fabrication involves only simple film deposition. This thermal camouflage technology, based on phase-changing material, can be applied to dynamically control the thermal emission in fundamental science and significantly benefit a number of applications, including thermal camouflage and heat management for outer space.In this study, a thermal camouflage device consisting of a Ge−4 Pa and 0.1 Pa separately, to maintain the deposition rate at 0.53 nm s−1.To fabricate GST-Au devices, a 100 nm-thick Au film and subsequently a 350 nm GST film are deposited on a silicon substrate by high vacuum magnetron sputtering under room temperature. Both the GST thickness and the metallic material have been optimized to achieve a large camouflage temperature range  is then spun onto the Au film and annealed for 5 min at 105 °C. The photoresist is exposed to define a checkerboard pattern and a ZJU pattern using a double-sided mask aligner system (MA6–BSA). The photoresist is then developed in 1:6 AR 300-26/ Deionized (DI) water followed by rinsing in DI water. After development, a 350 nm-thick GST film is then deposited onto the sample by magnetron sputtering. The spatially resolved GST-Au devices are created after lift-off by ultrasonic processing in acetone for 1 min.The infrared images are recorded by an infrared camera (FLIR S65). The spectral range of the infrared camera is between 7.5 μm and 13 μm. The spatial resolution of the infrared camera is 500 μm. The spectral radiance is measured by a Fourier transform infrared spectrometer (FTIR) with a room-temperature doped triglycine sulfate detector.The finite-difference time-domain method  is used to compute the optical responses of the MIM thermal emitter. The relative permittivity of gold is obtained from Palik’s handbook. The relative permittivities of GST (2.5 µm–15 µm) are obtained experimentally from the fabricated GST films  of an object recorded in infrared images is not equal to its real temperature (T) unless the object is a blackbody. The radiation temperature is related to several factors, which are included in the following equation:Tris the radiation temperature detected by an infrared (IR) camera, C is the specific constant of the IR camera, Pobj is the emitted power of the object detected by the IR camera, Iobj is the spectral radiance of the object, εobj is the emissivity of the object, and  is the spectral range of the IR camera (FLIR S65: 7.5 μm–13 μm). The radiation temperature of the object is directly related to the emitted power of the object Pobj, which is determined by the emissivity of the object, the real temperature of the object, and the spectral range of the IR camera. Most natural environments, such as bare soil (emissivity ε = 0.91), grass (ε = 0.95), and shrub (ε = 0.98), have emissivity close to a blackbody. Furthermore, the temperature of the environmental background keeps changing while the temperature of the object, such as a vehicle, remains fixed under most circumstances. This makes the direct camouflage of a fixed object in a varying background difficult. By covering the object with camouflage coating with tunable emissivity, the radiation temperature of the object can be controlled to match that of the background so that an infrared camera cannot distinguish the object from the background. In this study, thermal camouflage is explored in the situation in which the object temperature is fixed at 60 °C and the background temperature is changed from 30 °C to 50 °C, to mimic the situation of an object at varying background temperatures. Thermal camouflage is not investigated at background temperatures below 30 °C in this study, as this would necessitate an additional cooling system.The radiation temperature (T1 to T3 (T3 > T2 > T1), the background color changes from blue (cold: a low radiation temperature) to red (hot: a high radiation temperature) in the thermal images. The surface of the tank’s upper body at a fixed temperature T0 (T0 > T3) can be made to blend into the background by controlling the thermal emission based on different GST phases. The as-deposited GST is in the amorphous phase (termed aGST) with a disordered atomic distribution. Intermediate GST (termed iGST) and crystalline GST (termed cGST) with a well-organized atomic distribution are obtained by annealing it at 200 °C on a hot plate for less than and greater than 60 s, respectively  patterns of GST films with different annealing time  is shown in Fig. SThe experimental setup is shown in Fig. Infrared images are recorded to show the ability to achieve thermal camouflage at different background temperatures. When the background temperature is 30 °C, the radiation temperatures of both the object and the background are approximately 30 °C, and the object can hardly be distinguished from the background in the infrared image Fig. . To mimiThe measured spectral radiance at different temperatures of the background (termed BG) and different annealing time of the object (termed OB) is shown in Fig. P (Pmax − Pmin) is the difference between the emitted power of the cGST-Au and aGST-Au devices. If the emitted power of the background is in the range from Pmin to Pmax, then thermal camouflage can be achieved by controlling the GST phase to match the emitted power of the GST-Au device with that of the background. The temperature of the background at which its emitted power is the same as Pmax is Tmax. Similarly, the temperature of the background Tmin corresponds to Pmin. The background camouflage temperature range ΔT is the difference between Tmin and Tmax. The object can be camouflaged for a larger background temperature range by simply increasing the object temperature, as shown in Fig. SThe emitted powers of the background, aGST-Au device, and cGST-Au device are shown in Fig. The robustness of thermal camouflage to the observation angle over a large range from 0° to 60° is presented in Fig. The emissivities of GST-Au devices at different observation angles are calculated to validate the robustness of the thermal camouflage over different observation angles. The infrared camera receives the thermal emission of both Transverse Electric (TE) and Transverse Magnetic (TM) polarizations. Therefore, we calculate the average emissivities of TM and TE polarizations for aGST-Au and cGST-Au devices, which are shown in Fig. w = 1 mm and w = 500 µm), the smallest squares cannot be clearly identified from the background  before annealing. This is because both the GST and the metallic background have a low radiation temperature. When the annealing time is increased gradually, the GST squares (red color) become prominent and can be distinguished from the metallic background (blue color) as the radiation temperature of the GST squares increases. When the square pixel size w is reduced to 500 µm, which is close to our infrared camera’s spatial resolution, the contrast of the adjacent areas decreases, but the squares can still be identified from the background. For the ZJU pattern, it can be identified even though the line width of the ZJU pattern is 100 μm. For the checkerboard pattern, the spatial camouflage resolution is limited by the resolution of the infrared camera. However, this limit is broken in case of the ZJU pattern, as it has much less disturbance from adjacent GST structures. Therefore, it can be concluded that the spatial camouflage resolution is limited by the surrounding structures and the infrared camera resolution. The two blocks in Fig. To explore the camouflage limit for microscopic objects, the spatial emissivities of patterned GST-Au devices with different GST phases are studied further. The spatial shaping of the GST-Au devices’ emissivities can encode spatial information in infrared images by patterning the top layer GST. Spatially resolved infrared images of the checkerboard pattern and ZJU (abbreviated form of Zhejiang University) pattern are shown in Fig. Ts − Tb) between the GST film and the metallic background for different annealing time are shown in Fig. Ts and Tb are calculated by averaging the radiation temperature of ten arbitrarily selected points from GST and the metal background, respectively. The radiation temperature difference in the checkerboard pattern (w = 1 mm) increases from near 1 °C to 27 °C when the annealing time is increased from 0 s to 1000 s. For the square pixel size 500 µm, the radiation temperature difference can only increase up to 15 °C from 1 °C when the annealing time is increased from 0 s to 1000 s. The lower value of the radiation temperature difference for this checkerboard (w = 500 μm) can be attributed to the fact that the thermal camera spatial resolution is close to the size of the smallest GST squares. This leads to the lower average radiation temperature of the GST squares, whereas the average radiation temperature for the metal background is increased. The radiation temperature difference for the ZJU pattern increases up to approximately 6 °C from less than 1 °C when the annealing time of GST is increased from 0 s to 1000 s. The radiation temperature difference in the ZJU pattern is far less than that in the checkerboard pattern, because the ZJU line width (100 μm) is much less than the infrared camera’s spatial resolution (500 μm), and thermal emission from the ZJU line is averaged along with the adjacent low-emission metal background. This patterned GST on metal film can also be used to carry encoded spatial information, which can later be decoded using a thermal camera.The radiation temperature differences . The thermal camouflage is robust over a large range of observation angles, from 0° to 60°. Furthermore, it has been experimentally demonstrated that GST-Au devices with a top layer of patterned GST can encode spatial information, which can later be decoded using a thermal camera. This device also has the potential to work in a background changing from a high temperature to a low temperature by controlling the phase transition of GST from crystalline to amorphous (reamorphization). This reamorphization can be achieved by laser pulses (fs or ns)Supporting information